ABSTRACT the various tissues. Key words: Petroleum,

ABSTRACTThis study was carried out to assess the antioxidant  status of rats feddiet  incorporating catfish contaminatedwith crude petroleum  oil treatedwith  Monodoramyrstica extracts.

Thirty albino rats of weight 180 to 200 g were used forthe experiment and they were divided into six groups of five rats each.  Thegrouping were as follows, group  1:  control, group 2: rats were fed crudepetroleum oil contaminated catfish diet (CPO-CCD) only, group 3: CPO-CCD plustween 80, group 4, 5, and 6 were given CPO-CCD and treated with  M. myristica water extract  (MWE), M.myristica ethanol extract  (MEE) and M.

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myristica diethyl ether  extract (MDEE). The experiment lasted four weeks. The results showed significant(p<0.05) decreased in  blood reducedglutathione (GSH), blood oxidised glutathione (GSSG), superoxide dismutase(SOD), catalase (CAT) and increase malondialdehyde (MDA) level in the liver, kidney and brain rats fedCPO-CCD only and CPO-CCD + tween 80 when compared to the control.Administration of MWE, MEE and MDEE to the rats fed CPO-CCD significantly (p<0.05) increase the levelof  blood GSH, blood  GSSG, SOD, CAT and decrease MDA level in theliver, kidney and brain  when comparedwith the CPO-CCD only and CPO-CCD + Tween 80. No significant difference was observed in the  blood GSH:GSSG ratio and brain GSH level  in all the experimental groups.

Inconclusion, M. myristica  extracts exhibited  beneficial effect by improvement  of the antioxidant status and showed to evadethe oxidative insult elicited by the CPO-CCD intoxication and  in the various tissues.  Key words: Petroleum, Diet,  Antioxidants Indices,  Monodora MyrsticaINTRODUCTIONExposure tocrude oil pollution  leads to formationof free radicals (Won et al., 2016). Free radical is the most common reactiveoxygen species in human (Georgewill and Nwankwoala, 2008). Once polyaromantichydrocarbons enter the body of a living organism, each are metabolized to formhighly reactive molecules such as diol epoxides that are polyromantic hydrocarbonsintermediate metabolites that causes oxidative stress (Ekperusi and  Aigbodion, 2015; Penning, 2014).

Reducedglutathione is a multifunctional intracellular non-enzymatic antioxidant whichis well known to be the major thiol-disulphide redox buffer of the cell(Swaran, 2009). Oxidized glutathione is accumulated inside the cells and theratio of GSH/GSSG is a good measure of oxidative stress of an organisms. Superoxide dismutase is an enzyme that alternatelycatalyzes the dismutation of the superoxide radical into either ordinarymolecular oxygen or hydrogen peroxide (Hayyan et al.

, 2016). Catalase is also an enzymes. The name catalase wasgiven to the enzyme owing to its catalatic action on the hydrogen peroxide.

Antioxidant enzymes can beinactivated by lipid peroxides (Alantary et al., 2014; Hamza and Al-Harbi,2015). The subchronic exposure ofrats  to crude oil, decreased tissuescatalases activities (Nwaogu et al., 2011).

Several  studies have shown that, direct exposure tocrude oil can disrupt antioxidant status in serum, brain, liver, and kidney(Adedara  et al.,2012; Ebokaiwe andFarombi, 2016)Monodora myristica  is a spice commonly consumed in the NigerDelta part of Nigeria especially by the Itsekiri, Urhobo and Ndokwa  people of Delta State. The seeds of  M.myristica  have  attractive small,  possess antioxidant properties and can beused in pharmaceutical industries (Talalaji, 1999).  The present study aimed to assess the effectof M.

myristica  extracts in rats given  diet incorporating catfish  pollutedwith crude petroleum  oil by evaluating  someantioxidant  status such as  reduced and oxidised GSH, CAT, SOD and lipidperoxidation  level.   MATERIALS AND METHODSPreparation of the Spice (Monodora myristica) Extracts M. myristica was obtained  from Obiaruku  main market, Ukwuani Local Government Area(LGA), Delta State  and then lateridentified at the Department of Botany, Delta State  University, Abraka, Delta State. The spicewas briefly sun-dried to constant weight for two weeks and then crushed intofine particles using blender for at high speed.

One hundred grams of thepowdered spice was extracted with 500 ml of the respective solvent (hot water(60?C), ethanol (95 % v/v), and diethyl ether, 95 % v/v) and allowed to standfor 48 hrs. The mixture was then filtered using a clean muslin cloth. The  filtrate was evaporated to dryness usingrotary evaporator attached to a vacuum pump. The extracts were stored in refrigerator (- 4 oC) untilrequired. Crude petroleum oil pollution and diet preparation  The crudepetroleum oil (CPO) was got from the Nigerian National Petroleum Cooperation(NNPC), refinery, Warri Delta State, Nigeria. Fifty  catfish (with length between 20-25 cm  and weight between  250-300 g) was got  from commercial farm, then acclimatized for 7days  for the experiment.

The catfish was divided into two groups; group 1 :control : contains twenty-five catfish which was cultured in plastic aquariawith 30 L borehole water for four weeks. Group 2:  also contain twenty-five which was culturedin plastic aquaria with 30 L borehole water and then  polluted with crude petroleum oil, 823.3 µl/Las described by Ikeogu et al. (2013) for four weeks.   At the end of the experimental period, thecatfish was harvested and the used in the preparation of  diet for the experimental rats  following the method described bySunmonu  and Oloyede (2007).  The catfish  were oven dried at 40°Cand  used as a source of  protein.

The diet for each group were  prepared by mixing known quantities ofsources of each food class comprising: protein (25 %), corn  starch (52 %), groundnut oil (4 %),maize  cob (4 %), granulated  refined sugar (10 %) and vitamin/mineralmixture (5 %). The food components were  mixed together and then madeinto pellets which was  feed rats. Experimental RatsThirty malealbino Wistar rats were  used for thestudy.

The rats were allowed to acclimatized for two weeks to suite the  laboratory condition. They  had free access to water and standard growersmash diet. The rats  used for the studywere in accordance to the  guide for careand use of laboratory animals (NIH, 1985). They were divided into six  groups of five  rats; group 1:  control, group 2: CPO-CCD only, group 3:  CPO-CCD plus  1 ml/kg b. wt. of 1 % tween 80, group 4: CPO-CCDplus 200 mg/kg b. wt.

of MWE, Group 5:  CPO-CCD plus 200 mg/kg b. wt. of MEEand group 6: CPO-CCD plus 200mg/kg b. wt. of MDEE.

Rats in group 1 to 6 received tap water daily throughoutthe experiment. The  administration ofthe CPO-CCD and extracts orallywas allowed  for four weeks.  Blood Collection and Preparation of Tissue HomogenateThe ratswere  sacrified after 24 hours fast onthe last day. The blood was collected by cardiac puncture using hypodermicsyringe and needle and then transferred to an anticoagulant tube and organswere harvested. One gram of various tissue (liver, kidney and brain) were  homogenized in 10 ml of normal saline andthen centrifuged at 2,500 revolution per minutes for 15 minutes to obtained thesupernatant  which was immediately usedfor biochemical analysis. BIOCHEMICALANALYSISEstimation of blood GSH/GSSG (Reduced/Oxidized glutathione) ratio.Blood GSH/GSSG was  estimated using theenzymatic method described by Tietze (1976).

GSSG Sample preparation Thirty microliters (30 ?L) of thiol-scavenging reagent(50 mg 1- methyl pyridinium trifluoromethane sulphonate)  to a micro-centrifuge tube and 100 ?L of wholeblood was carefully added to the  bottomof the centrifuge tube and mixed gently. GSSG sample 130 ?L was incubated atroom temperature for 5-10 minutes. Thereafter 270 ?L of ice-cold 5 % MPA wasadded  to the tube  and centrifuged at 1000 x g and 4°Cfor 10 minutes. The supernatant (50 ?L) was added to 700 ?L of assay buffer(phosphate buffer, 2M, pH 8) in a new micro-centrifuge tube, this was placedthe  on ice until used.  GSH Sample preparation Fifty microliter (50 ?L) of whole blood  was added to the bottom of a micro-centrifugetube and  then mixed. Thereafter 350 ?Lof ice-cold 5% MPA  was added to themicro-centrifuge tubes and centrifuged at 1000 x g  in  4°Cfor 10 minutes.

Then, 25 ?L of the supernatant was added to 1.5 mL of assaybuffer in a new micro-centrifuge tubes and this was placed on ice untilrequired.ProcedureTwo hundred microliters (200 ?L)  of samples  and blank were  added to 200 ?L of the DTNB solution in  respective test-tubes, then  200 ?L of the reductase solution (recombinantglutathione reductase)  was addedimmediately then mixed. These were allowed to incubate at room temperature for 5 minutes, then 200 ?L of  2 mg/mL NADPH solution  was added to the test tubes.  The absorbancewere read and recorded at 412 nm.

Standard curve Afterpreparing 1mM stock solution of  GSH/GSSG. Different concentrations  wereprepared and from each of the GSH/GSSG dilution, 200?l was taken and added into2300 ?l of 0.2 M phosphate buffer pH 7.6 then 500?l of 1mM DTNB was added,these five mixtures were well shaken and incubated for five minutes.

Afterthe  incubation  period, absorbance of each mixture wasrecorded at  412 nm.5,5-dithiobis-2-nitrobenzoic acid (DTNB) blank was prepared by adding 500?l  DTNB to 2500 ?l phosphate buffer pH7.6.

Absorbance of DTNB was also taken at 412 nm. The real absorbance of eachmixture was obtained by subtracting  absorbance of DTBN blank from absorbance ofeach of the mixture. The concentration of GSSG is much lower in thereaction mixture compared to GSH,standard calibration  curve was plotted separately,  0, 0.50,0.75, 1.0, and 1.50 ?M  GSSG,  and 0, 1.

0,1.5, 2.0, and 3.0 ?M GSHConcentration of GSH/GSSG ratio (units/ml)  =   GSH-2GSSG    GSSG Estimation of tissue reduced glutathione The  reduced glutathione  concentration in the  liver, kidney and brain were  estimated using the method of Ellman (1959). Procedure: To 0.5 ml oftissue homogenate was added 2 ml 10% trichloroacetic acid and centrifuged. Onemilliliters (1ml) of supernatant was treated with 0.5 ml of Ellman’s reagentand 3 ml of phosphate buffer.

The colour developed was read at 412 nm. A seriesof standard were treated in similar manner along with a blank containing 3.5 mlof buffer.Determination of superoxide dismutase activity The activityof  SOD in the  liver, kidney and brainwere assayed using the method of Misra and Fridovich (1972). Procedure:The assay wascarried out by adding 0.

2 ml of the supernatant to 2.5 ml of 0.05 M carbonatebuffer, pH 10.2. The reaction was started by addition of 0.3 ml freshlyprepared epinephrine as the substrate to the buffer supernatant mixture and wasquickly mixed by inversion. The reference cuvette contained 2.5 ml of thebuffer; 0.

3 ml of the substrate and 0.2 ml of distilled water. The increase inabsorbance at 480 nm due to the adrenochrome formed was monitored every 30seconds for 120 seconds. Determination of Catalase Activity The method ofKaplan et al. (1972) was adopted forthe assay of liver, kidney and brain catalase activity.Procedure:Two milliliter(2 ml) of H2O2 was added to 1ml of sample  in the reaction cuvette. Absorbance was readat 360 nm for 70 seconds. The reference cuvette contained 2 ml H2O2and 1ml of water.

The disappearance of hydrogen peroxide was calculated usingthe Molar extinction  co- efficient, ? = 39.4 M-1 cm-1.Determination of Lipid Peroxidation Lipidperoxidation in form of malondialdehyde (MDA) were determined in the  liver, kidney and brain by using the methodof Buege and Aust (1978). Procedure:One millilitreof the sample  was added to 2 ml of  TCA-TBA-HCL reagent 0.

37% Thioarbituric acid(TBA), 15% Tricarcoxylic acid (TCA) and 0.24 N Hydrochloric acid (HCl) (1:1:1 ratio).The tube was stoppered  loosely andimmersed in boiling water for 15 minutes and swirled slightly at intervals. The mixture was cooled andcentrifuged for 10 minutes at 5000 g. The absorbance was read at 532 nm usingthe reagent blank. Lipid peroxidation in units/g of wet tissue wascalculated  with a molar extinctionco-efficient of 1.56 x 105M-1STATISTICAL ANALYSISThe  data obtained and results were expressed as mean ±SD. The significant differencesbetween groups were analyzed using  oneway analysis of variance (ANOVA) and least significant difference  (LSD).

The SPSS-PC programme package (version17.0) were used for statistical analysis. A significant threshold of p< 0.05was regarded statistically significance between the test and control group forthe analysis.

 RESULTS Table 1: Blood GSH, GSSG and GSH:GSSG ratio  of rats fed CPO-CCD   treated with extracts of M. myristica Groups Blood     GSH (units/ml) Blood             GSSG (units/ml) Blood GSH:GSSG ratio 1: Control  1.81±0.04 a 0.72±0.03 a 0.99±0.

02 a 2: CPO-CCD only  0.42±0.02 b 0.11±0.

07 b 0.80±0.05 a 3: CPO-CCD + Tween 80 0.42±0.

06 b 0.12±0.05 b 0.83±0.04 a 4: CPO-CCD + MWE 1.

09±0.05 a 0.53±0.01 a 1.00±0.

01 a 5: CPO-CCD + MEE 1.41±0.32 a 0.

56±0.01 a 1.01±0.02 a 6: CPO-CCD + MDEE 1.

63±0.10 a 0.61±0.

02 a 1.00±0.01 a Values are givenin mean ± SD. n=5.

Mean values with different superscript letter in the samecolumn differ significantly at p<0.05.  Table 2:  GSHlevel in the liver,  kidney andbrain  of rats fed CPO-CCD   treated with extracts of M. myristica   GSH  (units/g wet tissue) Groups Liver Kidney Brain 1: Control  7.19±0.99 a 6.54±0.

05 a 3.33±0.47 a 2: CPO-CCD only  3.36±0.94 b 2.24±0.79 b 1.

01±0.05 a 3: CPO-CCD + Tween 80 2.34±0.73 b 1.76±0.

57 b 2.81±0.08 a 4: CPO-CCD + MWE 3.46±0.54 b 3.31±0.

66 b 2.36±0.16 a 5: CPO-CCD + MEE 3.96±0.

09 b 4.29±0.19 b 2.20±0.16 a 6: CPO-CCD + MDEE 6.

25±0.58 a 5.31±0.29 a, b 2.64±0.08 a Valuesare given in mean ± SD.

n=5. Mean values with different superscript letter inthe same column differ significantly at p<0.05.  Table 3: Changes in superoxide dismutase activity in the liver, kidney and brainof rats fed CPO-CCD   treated with differentextracts of M. myristica   SOD (units/g wet tissue) Groups Liver Kidney Brain 1: Control  89.41±19.

16 a 86.41±14.28 a 73.50±6.52 a 2: CPO-CCD only  57.36±6.12 b 55.

07±8.00 b 40.28±3.15 b 3: CPO-CCD + Tween 80 56.07±14.40 b 54.46±3.03 b 40.

90±2.67 b 4: CPO-CCD + MWE 66.43±6.98 c 65.

29±6.29 c 50.48±4.33 c 5: CPO-CCD + MEE 78.11±6.77 d 74.

50±3.38 d 67.32±2.90 d 6: CPO-CCD + MDEE 85.47±3.

58 a 82.41±1.42 a 70.23±5.21 a Values are givenin mean ± SD. n=5. Mean values with different superscript letter in the samecolumn differ significantly at p<0.

05. Table 4: Changes in catalase activity in the liver, kidney and brain of rats fedCPO-CCD   treated with extracts of M. myristica.   CAT  (units/g wet tissue) Groups Liver Kidney Brain 1: Control  74.28±10.19 a 70.18±8.

65 a 64.26±5.94 a 2: CPO-CCD only  46.53±12.62 b 40.31±7.62 b 35.

71±3.92 b 3: CPO-CCD + Tween 80 46.16±512 b 41.15±2.95 b 36.27±4.

37 b 4: CPO-CCD + MWE 55.31±10.19 c 52.

28±3.47 c 45.49±3.64 c 5: CPO-CCD + MEE 61.47±11.53 d 60.74±5.42 d 50.

39±5.99 d 6: CPO-CCD + MDEE 72.20±5.65 a 68.32±3.

57 a 61.17±2.38 a Valuesare given in mean ± SD. n=5. Mean values with different superscript letter inthe same column differ significantly at p<0.

05.  Figure 1: MDA  level in the liver,  kidney and brain  of rats fed CPO-CCD treated with differentextracts of M. myristica.

Bars represent mean values from five rats in each group. For each organs,bars with different superscript letter in the same column differ significantlyat p<0.05.  RESULT AND DISCUSSION Alterations of blood GSH,GSSG and GSH:GSSG ratio of rats fed CPO-CCD  treated with different extracts of M.myristica are shown in Table 1. Significant (p<0.05) decrease level ofblood GSH and GSSG  were observed in ratsfed CPO-CCD only and CPO-CCD + tween 80 when compare with control.

Treatment ofrats fed CPO-CCD with MWE, MEE and MDEE CPO-CCD significantly (p<0.05)increase the level of blood GSH and GSSG. The reduction in  GSH and GSSGlevels  could be a compensatory mechanism by which the ratsfed the formulated feed mixed with crude oil contaminated catfish to overcomethe effect of the oxidant stress caused by free radicals produced by crudepetroleum oil. The is in linewith the findings of Shang et al. (2016), indicating that the decreased GSH to GSSGratio in the blood showed  that oxidativestress occurred in the distant organs and systemically upon crude petroleuminduced nephrotoxicity. No significantdifference were observed in the blood GSH, GSSG and GSH : GSSG ratio levelswhen rats fed CPO-CCD only were compared rats with fed CPO-CCD + tween 80, these results may be considered as  pathological evidences to confirm thenontoxic effect  of tween 80 aspreviously reported (Rowe, 2009). The level of GSH, SOD andCAT activity in the liver, kidney and brain of rats fed CPO-CCD treated withextracts of M.

myristica are shown inTable 2, 3 and 4 respectively. Rats fed with CPO-CCD only  and CPO-CCD + Tween 80  showed significant (p<0.05) decreased inSOD and CAT activity  in the liver,kidney and brain when compared to the control. Administration of MWE, MEE andMDEE to CPO-CCD rats  significantly(p<0.05) increased level of SOD and CAT in the liver, kidney and brain whencompared with the CPO-CCD only and CPO-CCD + Tween 80 respectively.  No significant difference was seen in GSHlevel in the brain of all the experimental groups.  The decrease kidneyand liver GSH level in the rats fed CPO-CCD may be due to the decrease in theactivity of the hepatic glutamate-cysteine ligase (a key enzyme responsible forglutathione synthesis).

The depletion in brain GSH inCPO-CCD induced oxidative stress may leads to increased productions ofsuperoxide, hydroxyl radicals, and H2O2, because there isno known enzymatic defense against hydroxyl radicals (Dringen, 2000), makingGSH the only compound capable of scavenging these radicals in the brain.   These findingsare in line with the study of Aoyama et al. (2008) which states that,when comparing the brain with other organs, the brain is especially vulnerableto oxidative stress. This is because it has lower SOD, CAT, and glutathioneperoxidase; GPx activities, while it contains an abundance of lipids withunsaturated fatty acids that are targets of lipid peroxidation (Dringen, 2000).Furthermore, the brain GSH concentration is lower than those of the liver andkidney (Aoyama et al.

, 2008). The detoxification mechanisms promoted by enhanced glutathioneproduction indicates the protective effects of MDEE, MEE and MWE. This also might be the reason for therestoration of other antioxidant enzymes (SOD and CAT).   The marked reduction in SOD activity of rats fed CPO-CCD  and the enhanced SOD activity  when M.myristica extracts was administered were in agreement with other studies(Nwaogu et al., 2011; Sunmonu and Oloyede, 2007). The inhibition of CATactivity during CPO-CCD induced toxicity  may be due to the increased generation ofreactive free radicals, which can lead to oxidative stress in the cells. Theadministration of MDEE, MEE and MWE inversed the catalase activity in theliver, kidney and brain tissues and thus enhance the antioxidant defenseagainst  ROS.

This findings are  in collaboration with Oyinloye et al.(2016)who reported that M. myristica aqueous extract prevent lipid peroxidation andreplenish hepatic antioxidant enzymes against cadmium induced  liver tissue damage. Figure 1, showed the levelof MDA; malondialdehyde (end product of membrane lipid peroxidation) inthe  tissues (liver, kidney and brain)ofrats fed CPO-CCD treated with extracts of M.myristica. MDA level in  therespective tissues were significantly (p<0.05) increased in rats fed  CPO-CCD only and CPO-CCD + tween 80 whencompared with the control rats.

  However, treatment of rats fed CPO-CCD with the different extractssignificantly decreased the level of MDA as compare with that of the control inthe respective tissues. The excessive ROS generated during crude petroleum  oil toxicity rapidlyreact with lipid membranes and thus initiates the lipid peroxidation chainreaction, resulting in lipid peroxyl radicals’ formation (Ita  and Edagha, 2016; Ujowundu et al., 2012; Nwaogu et al., 2011).

Theelevation of lipid peroxidation caused by rats administered crude petroleum oilhas been previously reported (Sunmonu and Oloyede, 2007), which is in line withthe results obtained in this study.  Inthe present study, the lower MDA levels in  the  tissues of rats fed CPO-CCD plus M. myristica  extracts, apparently indicating theanti-oxidative protective role of  M. myristica  extracts against CPO-CCD induced damage oncell membranes. Moreover, MDEE revealed a strong inhibitory ability towardslipid peroxidationas compared with MEE and MWE.

   CONCLUSIONThe results of this study showed that M. myristica  extracts  rescued the CPO-CCD induced tissuesdamage/lipid peroxidation and improvement of antioxidant status owing to its free radical scavenging properties.

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